G Activation of Src Induces Caveolae-mediated Endocytosis in Endothelial Cells*

Caveolae-mediated endocytosis in endothelial cells is stimulated by the binding of albumin to gp60, a specific albumin-binding protein localized in caveolae. The activation of gp60 induces its cell surface clustering and association with caveolin-1, the caveolar-scaffolding protein. This interaction leads to Gi-induced Src kinase activation, which in turn signals dynamin2-mediated fission and directed migration of caveolaederived vesicles from apical to basal membrane. In this study, we investigated the possible role of the G heterodimer in signaling Gi-induced Src activation and subsequent caveolae-mediated endocytosis. We observed using rat lung microvascular endothelial cells that expression of the C terminus of -adrenergic receptor kinase (ctARK), an inhibitor G signaling, prevented gp60-dependent Src activation as well as caveolae-mediated endocytosis and transcellular transport of albumin and uptake of cholera toxin subunit B, a specific marker of caveolae internalization. Expression of ctARK also prevented Src-mediated tyrosine phosphorylation of caveolin-1 and dynamin-2 and the resultant phosphorylation-dependent association of dynamin-2 and caveolin-1. Also, the direct activation of G using a specific cell-permeant activating peptide (myristoylated-SIRKALNILGYPDYD) simulated the effects of gp60 in inducing Src activation, caveolin-1, and dynamin-2 phosphorylation as well as caveolae-mediated endocytosis of cholera toxin subunit B. The myristoylated-SIRKALNILGYPDYD peptide-induced responses were inhibited by the expression of ctARK. Taken together, our results demonstrate that G activation of Src signals caveolaemediated endocytosis and transendothelial albumin transport via transcytosis.